Sustained release formulations of water soluble peptides

ABSTRACT

The invention discloses microparticles comprising a polypeptide, preferably somatostatin or an analog or derivative thereof, more preferably octreotide, in a polymeric matrix, preferably poly(lactide-co-glycolide) glucose. The invention also discloses sustained release formulations containing said microparticles and the use of said formulations in treating acromegaly and breast cancer.

This is a division of application Ser. No., 07/643,880, filed Jan. 18,1991, now U.S. Pat. No. 5,538,739, which in turn is acontinuation-in-part of application Ser. No. 07/411,347, filed Sep. 22,1989, which in turn is a continuation-in-part of application Ser. No.07/377,023, filed Ju. 7, 1989, the latter two of which are nowabandoned.

BACKGROUND OF THE INVENTION

This invention relates to sustained release (depot) formulations ofdrugs in particular water soluble peptides, e.g. somatostatin orsomatostatin analogs, such as octreotide, in a biodegradable andbiocompatible polymeric carrier, e.g. a matrix or a coating, e.g. in theform of a implant or preferably a microparticle (also known as amicrocapsule or a microsphere).

The invention also relates to such formulations, showing satisfactorypeptide release profiles over a particular period of time.

Peptide drugs often show after oral or parenteral administration a poorbioavailability in the blood, e.g. due to their short biologicalhalf-lives caused by their metabolic instability. If orally or nasallyadministered they additionally often show a Door resorption through themucuous membranes. A therapeutically relevant blood level over anextended period of time is difficult to achieve.

The parenteral administration of peptide drugs as a depot formulation ina biodegradable polymer, e.g. as microparticles or implants, has beenproposed enabling their sustained release after a residence time in thepolymer which protects the peptide against enzymatic and hydrolyticinfluences of the biological media.

Although some parenteral depot formulations of peptide drugs in apolymer in the form of microparticles or an implant, are known,satisfactory peptide release profiles are in practice only obtained invery few cases. Special measures must be taken to achieve a continuouspeptide release for a therapeutically active drug serum level and ifdesired avoiding too high drug serum concentrations, which causeundesired pharmacological side reactions.

The peptide drug release pattern is dependent on numerous factors, e.g.the type of the peptide, and e.g. whether it is present in its free orin another form, e.g. salt form, which may influence its watersolubility. Another important factor is the choice of polymer, from theextended list of possibilities which have been described in theliterature.

Each polymer type has its characteristic biological degradation rate.Free carboxyl groups may be formed which contribute to the pH value inthe polymer and thus additionally influence the water solubility of thepeptide and thus its release pattern.

Other factors, which may influence the release pattern of the depotformulation, are the drug loading of its polymeric carrier, the mannerof its distribution in the polymer, the particle size and, in case of animplant, additionally its shape. Further is the site of the formulationin the body of influence.

Until now no somatostatin composition in sustained release form forparenteral administration has reached the market, perhaps because nocomposition exhibiting a satisfactory serum level profile could beobtained.

DESCRIPTION OF THE PRIOR ART

Polymer formulations with drugs which are designed to give prolonged ordelayed release of the drug are known in the art.

U.S. Pat. No. 3,773,919 discloses controlled drug release formulationsin which the drug, e.g. a water soluble peptide drug is dispersed in abiodegradable and biocompatible linear polylactide orpolylactide-co-glycolide polymer. However, no drug release patterns havebeen described and there is no reference to a somatostatin. U.S. Pat.No. 4,293,539 describes anti-bacterial formulations in microparticleform.

U.S. Pat. No. 4,675,189 describes sustained release formulations of theLHRH analog decapeptide nafareline and analogous LHRH congeners inpolylactide-co-glycolide polymers. No release pattern has beendescribed.

T. Chang, J. Bioeng., Vol.1, pp.25-32, 1976 described prolonged releaseof biologicals, enzymes and vaccines from microparticles.

Polymers/copolymers of lactic acid and lactide/glycolide copolymers andrelated compositions for use in surgical applications and for sustainedrelease and biodegradation have been reported in U.S. Pat. Nos.3,991,776; 4,076,798 and 4,118,470.

European patent application 0 203 031 describes a series of somatostatinoctapeptide analogs, e.g. Compound RC-160 having the formula:

    D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Trp-NH.sub.2,

having a bridge between the -Cys- moieties, in columns 15-16.

The possibility of the somatostatins being microencapsulated withpolylactide-co-glycolide polymer has been mentioned in claim 18, but noinstructions have been disclosed how to obtain a continuoustherapeutically active serum level.

U.S. Pat. No. 4,011,312 describes that a continuous release of anantimicrobial drug, e.g. the water soluble polymyxin B from apolylactide-co-glycolide matrix of a low molecular weight (below 2000)and a relatively high glycolide content in the form of an implant, canbe obtained, when the implant is inserted into the teat canal of a cow.The drug is released within a short period of time, due to the highglycolide content and the low molecular weight of the polymer, whichboth stimulate a quick polymer biodegradation and thus a correspondingquick release of the drug. A relatively high drug loading contentadditionally contributes to a quick drug release. No somatostatins andno drug release patterns have been described.

European Patent No. 58481 discloses that a continuous release of a watersoluble peptide from a polylactide polymer implant is stimulated bylowering the molecular weight of at least a part of the polymermolecules, by introducing glycolide units into the polymer molecule, byincreasing the block polymer character of the polymer whenpolylactide-co-glycolide molecules are used, by increasing the drugloading content of the polymer matrix and by enlarging the surface ofthe implant.

Although somatostatins are mentioned as water soluble peptides, nosomatostatin release profiles have been described and no indication hasbeen given how to combine all these parameters to obtain e.g. acontinuous somatostatin serum level over at least one week, e.g. onemonth.

European Patent No. 92918 describes that a continuous release ofpeptides, preferably of hydrophilic peptides, over an extended period oftime can be obtained, when the peptide is incorporated in a conventionalhydrophobic polymer matrix, e.g. of a polylactide, which is made moreaccessible for water by introducing in its molecule a hydrophilic unit,e.g. of polyethyleneglycol, polyvinylalcohol, dextran orpolymethacrylamide. The hydrophilic contribution to the amphipathicpolymer is given all the ethylene oxide groups in case of a polyethyleneglycol unit, by the free hydroxyl groups in the case of apolyvinylalcohol unit or of a dextran unit, and by the amide groups inthe case of a polymethyacrylamide unit. Due to the presence of thehydrophilic unit in the polymer molecules the implant will obtainhydrogel properties after the absorption of water. Somatostatin ismentioned as an hydrophilic peptide, but no release profile has beendescribed and no indication has been given, what type of polymer ispreferred for this peptide, and what molecular weight and how manyhydrophilic groups it should have.

GB 2,145,422 B describes that a sustained release of drugs of severaltypes, e.g. of vitamins, enzymes, antibiotics, antigens, can be obtainedover an extended period of time, when the drug is incorporated in animplant, e.g. of microparticle size, made of a polymer of a polyol, e.g.glucose or mannitol, having one or more, preferably at least 3,polylactide ester groups. The polylactide ester groups preferablycontain e.g. glycolide units.

No peptides, e.g. somatostatins, are mentioned as drugs and no serumdrug levels have been disclosed.

SUMMARY OF THE INVENTION

This invention relates to sustained release formulations, e.g.microparticle formulations, of a drug, especially of a hormonally activewater-soluble somatostatin or a somatostatin analog such as octreotide,providing a satisfactory drug plasma level and, e.g. in a biodegradable,biocompatible polymer, e.g. in a encapsulating polymer matrix. Thepolymer matrix may be a synthetic or natural polymer.

The microparticles of this invention may be prepared by any conventionaltechnique, e.g. an organic phase separation technique, a spray dryingtechnique or a triple emulsion technique, wherein the polymer isprecipitated together with the drug, followed by hardening of theresulting product, when the phase separation or triple emulsiontechnique are used.

If desired the sustained release formulations may be in the form of animplant.

We have found an especially useful modification of the phase separationtechnique for preparing microparticles of any drug.

Accordingly the present invention also provides a process for theproduction of a microparticle comprising a drug in a biodegradable,biocompatible carrier which comprises the steps of:

a) dissolving the polymeric carrier material in an appropriate solvent,in which the drug compound is not soluble.

b) adding and dispersing a solution of the drug compound in anappropriate solvent, e.g. an alcohol, which is a non-solvent for thepolymer, in the solution of step a),

c) adding a phase inducing agent to the dispersion of step b), to inducemicroparticle formation,

d) adding the mixture of step c) to an oil-in-water emulsion to hardenthe microparticle, and

e) recovering the microparticle.

We have also found an especially useful modification of the tripleemulsion technique for preparing microparticles of any drug.

Accordingly the present invention provides:

A process for producing microparticles which comprises

(i) intensively mixing a water-in-oil emulsion formed from an aqueousmedium and a water-immiscible organic solvent containing in one phasethe drug and in the other a biodegradable, biocompatible polymer, withan excess of an aqueous medium containing an emulsifying substance or aprotective colloid to form a water-in-oil-in-water emulsion, withoutadding any drug retaining substance to the water-in-oil emulsion orapplying any intermediate viscosity increasing step,

(ii) desorbing the organic solvent therefrom, and

(iii) isolating and drying the resultant microparticles.

The present invention additionally provides the microparticles obtainedaccording to these processes.

The present invention also provides:

a) a sustained release formulation comprising a peptide drug compound ina 40/60 to 60/40 polylactide-co-glycolide ester of a polyol, the polyolunit chosen from the group of a (C₃₋₆)carbon chain containing alcoholhaving 3 to 6 hydroxyl groups and a mono- or di-saccharide, and theesterified polyol having at least 3 polylactide-co-glycolide chains.

b) A sustained release formulation comprising a peptide drug compoundchosen from the group of a calcitonin, lypressin or a somatostatin in a40/60 to 60/40 polylactide-co-glycolide polymer having linear chains ofa molecular weight M_(w) between 25,000 and 100,000, a polydispersityM_(w) /M_(n) between 1.2 and 2 in a concentration of from 0.2,preferably 2 to 10% of weight of the peptide drug compound therein.

c) A sustained release formulation comprising octreotide or a salt or aderivative thereof in a biodegradable, biocompatible polymeric carrier.

We have found that a novel salt of octreotide is the pamoate which isvery stable in such formulations.

The present invention accordingly provides (i) octreotide pamoate and(ii) a process for the production of octreotide pamoate which comprisesreacting octreotide with embonic acid (or a reactive derivativethereof).

Additionally the present invention provides:

A method of administering a peptide to a subject which comprisesadministering parenterally to a subject in need of such treatment adepot formulation as defined above, especially for the treatment ofacromegaly or breast cancer.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The drugs of use in the processes of the invention are preferably watersoluble drugs, e.g. peptides.

The peptides of use in the processes and formulations of this inventionmay be a calcitonin, such as salmon calcitonin, lypressin, and thenaturally occuring somatostatin and synthetic analogs thereof.

The naturally occuring somatostatin is one of the preferred compoundsand is a tetradecapeptide having the structure: ##STR1##

This hormone is produced by the hypothalmus gland as well as otherorgans, e.g. the GI tract, and mediates, together with GRF, q.v. theneuroregulation of pituitary growth hormone release. In addition toinhibition of GH release by the pituitary, somatostatin is a potentinhibitor of a number of systems, including central and peripheralneural, gastrointestinal and vascular smooth muscle. It also inhibitsthe release of insulin and glucagon.

The term "somatostatin" includes its analogues or derivatives thereof.By derivatives and analogues is understood straight-chain, bridged orcyclic polypeptides wherein one or more amino acid units have beenomitted and/or replaced by one or more other amino radical(s) of and/orwherein one or more functional groups have been replaced by one or moreother functional groups and/or one or more groups have been replaced byone or several other isosteric groups. In general, the term covers allmodified derivatives of a biologically active peptide which exhibit aqualitatively similar effect to that of the unmodified somatostatinpeptide.

Agonist analogs of somatostatin are thus useful in replacing naturalsomatostatin in its effect on regulation of physiologic functions.

Preferred known somatostatins are:

a) (D)Phe-Cys-Phe-(D)Trp-Lys-Thr-Cys-Thr-ol (Generic name Octreotide)

b) (D)Phe-Cys-Tyr-(D)Trp-Lys-Val-Cys-ThrNH₂

c) (D)Phe-Cys-Tyr-(D)Trp-Lys-Val-Cys-TrpNH₂

d) (D)Trp-Cys-Phe-(D)Trp-Lys-Thr-Cys-ThrNH₂

e) (D)Phe-Cys-Phe-(D)Trp-Lys-Thr-Cys-ThrNH₂

f) 3-(2-(Naphthyl)-(D)Ala-Cys-Tyr-(D)Trp-Lys-Val-Cys-ThrNH₂

g) (D)Phe-Cys-Tyr-(D)Trp-Lys-Val-Cys-β-Nal-NH₂

h) 3-(2-naphthyl)-Ala-Cys-Tyr-(D)Trp-Lys-Val-Cys-β-Nal-NH₂

i) (D)Phe-Cys-β-Nal-(D)Trp-Lys-Val-Cys-Thr-NH₂

wherein in each of compounds a) to i) there is a bridge between theamino acids marked with a * as indicated in the next formula.

Other preferred somatostatins are: ##STR2##

(See Vale et al., Metabolism, 27, Supp. 1, 139 (1978)).

    Asn-Phe-Phe-(D)Trp-Lys-Thr-Phe-Gaba

(See European Pat. Publication No. 1295 and Appln. No. 78 100 994.9).

    MeAla-Tyr-(D)Trp-Lys-Val-Phe

(See Verber et al., Life Sciences, 34, 1371-1378 (1984) and EuropeanPat. Appln. No. 82106205.6 (published as No. 70 021)) also known ascyclo (N-Me-Ala-Tyr-D-Trp-Lys-Val-Phe).

    NMePhe-His-(D)Trp-Lys-Val-Ala

(See R. F. Nutt et al., Klin. Wochenschr. (1986) 64 (Suppl. VII)

    H-Cys-His-His-Phe-Phe-(D)Trp-Lys-Thr-Phe-Thr-Ser-Cys-OH

(see EP-A-200,188).

    X-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-NH.sub.2

and

    X-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol

wherein X is a cationic anchor especially

    Ac-hArg(Et.sub.2)-Gly-Cys-Phe-D-Trp-Lys-Thr-Cys-NH.sub.2

(See EP 0363589A2)

wherein in the above mentioned amino acids there is a bridge between theamino acids marked with a *.

The contents of all the above publications including the specificcompounds are specifically incorporated herein by reference.

The term derivative includes also the corresponding derivatives bearinga sugar residue.

When somatostatins bear a sugar residue, this is preferably coupled to aN-terminal amino group and/or to at least one amino group present in apeptide side chain, more preferably to a N-terminal amino group. Suchcompounds and their preparation are disclosed, e.g. in WO 88/02756.

The term octreotide derivatives includes those including the moiety

    -D-Phe-Cys-Phe-DTrp-Lys-Thr-Cys-

having a bridge between the Cys residues.

Particularly preferred derivatives are N.sup.α -α-glucosyl-(1-4-deoxyfructosyl!-DPhe-Cys-Phe-DTrp-Lys-Thr-Cys-Thr-ol andN.sup.α - β-deoxyfructosyl-DPhe-Cys-Phe-DTrp-Lys-Thr-Cys-Thr-ol, eachhaving a bridge between the -Cys- moieties, preferably in acetate saltform and described in Examples 2 and 1 respectively of the abovementioned application.

The somatostatins may exist e.g. in free form, salt form or in the formof complexes thereof. Acid addition salts may be formed with e.g.organic acids, polymeric acids and inorganic acids. Acid addition saltsinclude e.g. the hydrochloride and acetates. Complexes are e.g. formedfrom somatostatins on addition of inorganic substances, e.g. inorganicsalts or hydroxides such as Ca- and Zn-salts and/or an addition ofpolymeric organic substances.

The acetate salt is a preferred salt for such formulations, especiallyfor microparticles leading to a reduced initial drug burst. The presentinvention also provides the pamoate salt, which is useful, particularlyfor implants and the process for its preparation.

The pamoate may be obtained in conventional manner, e.g. by reactingembonic acid (pamoic acid) with octreotide e.g. in free base form. Thereaction may be effected in a polar solvent, e.g. at room temperature.

The somatostatins are indicated for use in the treatment of disorderswherein long term application of the drug is envisaged, e.g. disorderswith an aetiology comprising or associated with excess GH-secretion,e.g. in the treatment of acromegaly, for use in the treatment ofgastrointestinal disorders, for example, in the treatment or prophylaxisof peptic ulcers, enterocutaneous and pancreaticocutaneous fistula,irritable bowel syndrome, dumping syndrome, watery diarrhea syndrome,acute pancreatitis and gastroenteropathic endocrine tumors (e.g.vipomas, GRFomas, glucagonomas, insulinomas, gastrinomas and carcinoidtumors) as well as gastro-intestinal bleeding, breast cancer andcomplications associated with diabetes.

The polymeric carrier may be prepared from biocompatible andbiodegradable polymers, such as linear polyesters, branched polyesterswhich are linear chains radiating from a polyol moiety, e.g. glucose.Other esters are those of polylactic acid, polyglycolic acid,polyhydroxybutyric acid, polycaprolactone, polyalkylene oxalate,polyalkylene glycol esters of acids of the Kreb's cycle, e.g. citricacid cycle and the like and copolymers thereof.

The preferred polymers of this invention are the linear polyesters, andthe branched chain polyesters. The linear polyesters may be preparedfrom the alphahydroxy carboxylic acids, e.g. lactic acid and glycolicacid, by the condensation of the lactone dimers, see for example U.S.Pat. No. 3,773,919.

Linear polylactide-co-glycolides which are preferably used according tothe invention conveniently have a molecular weight between 25,000 and100,000 and a polydispersibility Mw/Mn e.g. between 1.2 and 2.

The branched polyesters preferably used according to the invention maybe prepared using polyhydroxy compounds e.g. polyol e.g. glucose ormannitol as the initiator. These esters of a polyol are known anddescribed in GB 2,145,422 B. The polyol contains at least 3 hydroxygroups and has a molecular weight of up to 20,000, with at least 1,preferably at least 2, e.g. as a mean 3 of the hydroxy groups of thepolyol being in the form of ester groups, which contain poly-lactide orco-poly-lactide chains. Typically 0.2% glucose is used to initiatepolymerization. The structure of the branched polyesters is star shaped.The preferred polyester chains in the linear and star polymer compoundspreferably used according to the invention are copolymers of the alphacarboxylic acid moieties, lactic acid and glycolic acid, or of thelactone dimers. The molar ratios of lactide: glycolide is from about75:25 to 25:75, e.g. 60:40 to 40:60, with from 55:45 to 45:55, e.g.55:45 to 50:50 the most preferred.

The star polymers may be prepared by reacting a polyol with a lactideand preferably also a glycolide at an elevated temperature in thepresence of a catalyst, which makes a ring opening polymerizationfeasible.

We have found that an advantage of the star polymer type in theformulations of the present invention is, that its molecular weight canbe relatively high, giving physical stability, e.g. a certain hardness,to implants and to microparticles, which avoids their sticking together,although relatively short polylactide chains are present, leading to acontrollable biodegradation rate of the polymer ranging from severalweeks to one or two months and to a corresponding sustained release ofthe peptide, which make a depot formulation made therefrom suitable fore.g. a one month's release.

The star polymers preferably have a main molecular weight M_(w) in therange of from about 10,000 to 200,000, preferably 25,000 to 100,000,especially 35,000 to 60,000 and a polydispersity e.g. of from 1.7 to3.0, e.g. 2.0 to 2.5. The intrinsic viscosities of star polymers ofM_(w) 35,000 and M_(w) 60,000 are 0.36 resp. 0.51 dl/g in chloroform. Astar polymer having a M_(w) 52,000 has a viscosity of 0.475 dl/g inchloroform.

The terms microsphere, microcapsule and microparticle are considered tobe interchangeable with respect to the invention, and denote theencapsulation of the peptides by the polymer, preferably with thepeptide distributed throughout the polymer, which is then a matrix forthe peptide. In that case preferably the terms microsphere or moregenerally microparticle are used.

Using the phase separation technique of the present invention theformulations of this invention may be prepared for example by dissolvingthe polymeric carrier material in a solvent, which is a nonsolvent forthe peptide, following by the addition and dispersing a solution of thepeptide in the polymer-solvent composition. A phase inducer e.g. asilicone fluid is then added to induce encapsulation of the peptide bythe polymer.

The drug burst effect can be significantly reduced by in situprecipitation of ultra fine drug particules, by adding a drug solutionto the polymer solution prior to phase separation. The prior art methodinvolves adding dry particles directly to the polymer solution.

The therapeutic duration of peptide release can be increased byhardening/washing the microparticles with an emulsion of buffer/heptane.The prior art method involves a hardening step followed by either nosubsequent washing, or a separate aqueous washing step.

An emulsion of the type oil-in-water (=o/w) may be used to wash andharden the microspheres and remove non-encapsulated peptide. The washaids in the removal of non-encapsulated peptide from the surface of themicrospheres. The removal of excess peptide from the microspheresdiminishes the initial drug burst, which is characteristic of manyconventional encapsulation formulations. Thus, a more consistent drugdelivery over a period of time is possible with the present microsphereformulations.

The emulsion also aids, in the removal of residual polymer solvent andthe silicone fluid. The emulsion may be added to the polymer peptidemixture, or the mixture added to the emulsion. It is preferred that thepolymer peptide mixture be added to the emulsion.

The o/w emulsion may be prepared using a emulsifier such as sorbitanmono-oleate (Span 80 ICI Corp.) and the like, to form a stable emulsion.The emulsion may be buffered with a buffer which is non-detrimental tothe peptide and the polymer matrix material. The buffer may be from pH 2to 8 with a pH 4 preferred. The buffer may be prepared from acidicbuffers such as phosphate buffer, acetate buffer and the like. Wateralone may be substituted for the buffer.

Heptane, hexane and the like may be used as the organic phase of thebuffer.

The emulsion may contain dispersing agents such as silicone oil.

A preferred emulsion may comprise heptane, pH 4 phosphate buffer,silicone oil and sorbitan mono-oleate. When an initial drug release maybe desirable, a single non-solvent hardening step may be substituted forthe emulsion hardening. Heptane, hexane and the like, may be used as thesolvent.

Other alternatives to the o/w emulsion may be used for hardening themicrocapsules, such as:

Solvent plus emulsifier for hardening the microcapsules without washing;and solvent plus emulsifier for hardening followed by a separate washingstep.

The o/w emulsion may be used without the dispersing agent. Thedispersing agent, however, avoids aggregation of the dry particles ofmicrocapsules due to static electricity, and helps to reduce the levelof residual solvent.

Examples of the solvent for the polymer matrix material includemethylene chloride, chloroform, benzene, ethyl acetate, and the like.The peptide is preferably dissolved in an alcoholic solvent, e.g.methanol, which is miscible with the polymer solvent.

The phase inducers (coacervation agents) are solvents which are misciblewith the polymer-drug mixture, and cause the embryonic microcapsules toform prior to hardening; silicone oils are the preferred phase inducers.

The o/w emulsion may be prepared in a conventional manner using heptane,hexane and the like for the organic phase.

The microparticles of this invention may also be prepared by thegenerally known spray-drying procedure. According to this method thesomatostatin, or a solution of the peptide in an organic solvent, e.g.methanol, in water or in a buffer, e.g of pH 3-8 and a solution of thepolymer in an organic solvent, not miscible with the former one, e.g.methylene chloride, are thoroughly mixed.

The formed solution, suspension or emulsion is then sprayed in a streamof air, preferably of warm air. The generated microparticles arecollected, e.g. by a cyclon and if desired washed, e.g. in a buffersolution of e.g. pH 3.0 to 8.0 preferably of pH 4.0 or distilled waterand dried in a vacuum e.g. at a temperature of 20° to 40° C. The washingstep can be applied, if the particles exhibit a drug burst in vivo, andthe extent of the drug burst would be undesired. As a buffer an acetatebuffer can be used.

Microparticles can accordingly be obtained, exhibiting an improvedsomatostatin release profile in vivo.

The invention thus also relates to the microparticles prepared by thisprocess. The invention thus additionally provides a sustained releaseformulation prepared by mixing a somatostatin or a solution of asomatostatin in methanol or water or a buffer of pH 3-8 and a solutionof the polylactide-co-glycolide in methylene chloride and spraying theformed solution, emulsion or suspension of somatostatin in the polymersolution in a stream of warm air, collecting the microspheres andwashing them in a buffer solution of pH 3.0 to 8.0 or destilled waterand drying them in a vacuum at a temperature of from 20° to 40° C.Compared with microparticles, prepared according to the phase separationtechnique, they do not contain silicon oil, even not in traces, since nosilicon oil is used in the spray drying technique.

The formulations of the invention may also be prepared using atriple-emulsion procedure. In a typical technique, peptide e.g.octreotide is dissolved in a suitable solvent e.g. water and emulsifiedintensively into a solution of the polymer, e.g. 50/50poly(D,L-lactide-co-glycolide)glucose in a solvent, which is anon-solvent for the peptide, e.g. in methylene chloride. Examples of thesolvent for the polymer matrix material include methylene chloride,chloroform, benzene, ethyl acetate, and the like. The resultingwater/oil (w/o) emulsion is further emulsified into an excess of water,containing an emulsifying substance, e.g. an anionic or non-ionicsurfactant or lecithin or a protective colloid e.g. gelatine, dextrin,carboxymethylcellulose, polyvinylpyrrolidone or polyvinyl alcohol, whichprovides continuous generation of the triple (w/o/w) emulsion. Themicroparticles are formed by spontaneous precipitation of the polymerand hardened by evaporation of the organic solvent. Gelatine serves toprevent agglomeration of the microspheres. After sedimentation of themicroparticles the supernatant is decanted and the microparticles arewashed with water and then with acetate buffer. The microparticles arethen filtered and dried.

The peptide can also be dispersed directly in the polymer solution,thereafter the resulting suspension is mixed with the gelatinecontaining water phase.

The triple emulsion procedure is known from the U.S. Pat. No. 4,652,441.According to this patent in a first step a drug solution (1) in asolvent, e.g. somatostatin in water (Column 2, lines 31-32), isthoroughly mixed with an excess of a polylactide-co-glycolide solution(2) in another solvent, in which the first solvent is not soluble, e.g.methylene chloride, giving a water-in-oil type (w/o) emulsion (3) offine drug-containing droplets of (1) in solution (2).

In solution (1) is additionally dissolved a so-called drug-retainingsubstance (Column 1, line 31), e.g. gelatin, albumin, pectin, or agar.

In a second step, the viscosity of the inner phase (1) is increased byappropriate means, like heating, cooling, pH change, addition of metalions, or cross linking of e.g. gelatin with an aldehyde.

In a third step, an excess of water is thoroughly mixed with thew/o-emulsion (3), (Column 7, lines 52-54), leading to a w/o/w-typeternary-layer emulsion. In the excess of water a so-called emulsifyingagent may if desired be present (Column 7, line 56), chosen from thegroup of e.g. an anionic or nonionic surfactant or e.g. polyvinylpyrrolidone, polyvinyl alcohol or gelatine.

In a fourth step, the w/o/w-emulsion is subjected to "in-water drying",(line 52). This means that the organic solvent in the oil layer isdesorbed to generate microparticles.

The desorption is accomplished in a manner known per se (Column 8, lines3-5), e.g. by pressure decrease while stirring (Column 8, lines 5-7) ore.g. by blowing nitrogen gas through the oil layer (e.g. methylenechloride) (line 19).

The formed microparticles are recovered by centrifugation or filtration(lines 26-27) and the components which are not incorporated in thepolymer are removed by washing with water (line 29). If desired, themicroparticles are warmed under reduced pressure to achieve) betterremoval of water and of solvent (e.g. methylene chloride from themicroparticle wall (lines 30-32).

Whilst the above process is satisfactory for the production offormulations according to the invention, however, the so-calleddrug-retaining substance mentioned above, e.g. gelatine, albumin, pectinor agar, is still enclosed in the resultant microparticles.

We have now found that when the addition of the drug retaining substance(=in solution (1)) and the step of increasing the viscosity of the innerphase is avoided, and in the excess of water of the ternaryw/o/w-emulsion, the measure of adding an emulsifying substance or aprotective colloid, like gelatine is maintained, satisfactorymicroparticles can still be obtained. Additionally, the microparticlesdo not contain any drug retaining substance, and only a very smallquantity of methylene chloride.

Therefore the invention provides a process for the production ofmicroparticles prepared by intensively mixing:

a) a solution of a drug, preferably a somatostatin, especiallyoctreotide in an aqueous medium, preferably water or a buffer,preferably in a weight/volume ratio of 0.8 to 4.0 g 1 to 120 ml,especially 2.5/10 and in a buffer of pH 3-8, especially an acetatebuffer, and

b) a solution of a polymer, preferably a polylactide-co-glycolide, suchas mentioned above, in an organic solvent, not miscible with the aqueousmedium, e.g. methylene chloride, preferably in a weight/volume ratio of40 g/90 to 400 ml, especially 40/100, preferably in such a manner thatthe weight/weight ratio of the drug to the polymer is from 1/10 to 50,especially 1/16 and the volume/volume ratio of the aqueousmedium/organic solvent is 1/1.5 to 30, especially 1/10, intensivelymixing the w/o-emulsion of a) in b) together with

c) an excess of an aqueous medium, preferably water or a buffer, e.g. anacetate or phosphate buffer, preferably of a pH 3-8, containing anemulsifying substance or a protective colloid, preferably in aconcentration of 0.01 to 15.0%, particularly gelatine, especially in aconcentration of 0.1 to 3%, particularly 0.5% of weight, preferably at avolume/volume mixing speed ratio of ab)/c) of from 1/10 to 100,especially 1/40,

without adding any drug retaining substance to the water-in-oil emulsionor applying any intermediate viscosity increasing step, hardening theembryonic microparticles in the formed w/o/w-emulsion by desorption,preferably by evaporation, of the organic solvent, preferably methylenechloride, and by isolating, optionally washing and drying the generatedmicroparticles.

The invention also provides the process variant, in which the drug isdispersed directly in the polymer solution, whereafter the resultingdispersion is mixed with the gelatine containing water phase.

The invention also provides the microparticles produced by theseprocesses. Like microparticles prepared according to the spray dryingtechnique, they do not contain silicon oil. Compared with microparticlesprepared according to the known triple emulsion process type, they donot contain any amount of a protective colloid.

The sustained release formulations can also be made by other methodsknown per se, e.g.

if the peptide is stable enough for the production of an implant, byheating microparticles containing the peptide, e.g. a somatostatin in apolylactide-co-glycolide, especially such as described above or amixture thereof obtained-by mixing the peptide and the polymer, at atemperature of e.g. from 70° to 100° C. and extruding and cooling thecompact mass, after which the extrudate is cut and optionally washed anddried.

Conveniently the formulations according to the invention are producedunder aseptic conditions.

The formulations according to the invention may be utilized in depotform, e.g. injectable microspheres or implants.

They may be administered in conventional manner, e.g. subcutaneous orintramuscular injection, e.g. for indications known for the drugcontained therein.

The sustained release formulations containing octreotide may beadministered for all the known indications of the octreotide orderivatives thereof, e.g. those disclosed in GB 2,199,829 A pages 89-96,as well as for acromegaly and for breast cancer.

The microparticles of this invention may have a size range from about 1to 250 microns diameter, preferably 10 to 200, especially 10 to 130,e.g. 10 to 90 microns. Implants may be e.g. from about 1 to 10 cubic mm.The amount of drug i.e. peptide present in the formulation depends onthe desired daily release dosage and thus on the biodegradation rate ofthe encapsulating polymer. The exact amount of peptide may beascertained by bioavailability trials. The formulations may containpeptide in an amount from at least 0.2, preferably 0.5 to 20 per cent byweight relative to the polymeric matrix, preferably 2.0 to 10,especially 3.0 to 6% of weight.

The release time of the peptide from the microparticle may be from oneor two weeks to about 2 months.

Conveniently the sustained release formulation comprises a somatostatin,e.g. octreotide in a biodegradable biocompatible polymeric carrierwhich, when administered to a rat subcutaneously at a dosage of 10 mgsomatostatin per kg of animal body weight, exhibits a concentration of asomatostatin in the blood plasma of at least 0.3 ng/ml and preferablyless than 20 ng/ml during a 30 day term, or conveniently a 60 day'sterm.

Alternatively conveniently the sustained release formulation comprises asomatostatin, e.g. octreotide in a biodegradable biocompatible polymericcarrier, which, when administered to a rabbit intramuscularly at adosage of 5 mg per kg of body weight, exhibits a concentration of asomatostatin of at least 0.3 ng/ml during a 50 day's term andconveniently a concentration of at most 20 ng/ml.

Further preferred properties of the obtained somatostatin, e.g.octreotide containing depot formulations are, depending on the usedproduction processes:

    ______________________________________    Phase separation technique    Rabbit 5 mg of somatostatin/kg,    intramuscularly    retardation      (0-42 days)                                76%    average plasma level (cp, ideal)                     (0-42 days)                                4 ng/ml    AUC              (0-42 days)                                170 ng/ml × days    Spray drying technique:    Rat 10 mg of somatostatin/kg,    subcutaneously    retardation      (0-42 days)                                >75%    average plasma level (cp, ideal)                     (0-42 days)                                4-6 ng/ml    AUC              (0-42 days)                                170-210 ng/ml ×                                days    Rabbit 5 mg of somatostatin/kg,    intramuscularly    retardation      (0-43 days)                                >75%    average plasma level (cp, ideal)                     (0-43 days)                                4-6 ng/ml    AUC              (0-43 days)                                200-240 ng/ml ×                                days    Triple emulsion technique:    Rat 10 mg of somatostatin/kg,    subcutaneously    retardation      (0-42 days)                                >75%    average plasma level (cp, ideal)                     (0-42 days)                                4-6.5 ng/ml    AUC              (0-42 days)                                170-230 ng/ml ×                                days    Rabbit 5 mg of somatostatin/kg,    intramuscularly    retardation      (0-42/43 days)                                >74%    average plasma level (cp, ideal)                     (0-42/43 days)                                3.5-6.5 ng/ml    AUC              (0-42/43 days)                                160-270 ng/ml ×                                days    ______________________________________

The invention thus also provides somatostatin preferably octreotide andoctreotide analog compositions, having the following properties:

1. a retardation of at least 70%, preferably at least 74%, e.g. at least75%, 80%, 88% or at least 89% over a period of from 0 to 42 or 43 daysand/or

2. an average plasma level (C_(p).ideal) of 2.5-6.5, preferably 4-6.5ng/ml over a period of from 0 to 42 days, in the rat, when 10 mg ofsomatostatin is subcutaneously administered and/or an average plasmalevel of 3.5-6.5, e.g. 4-6.5 ng/ml over a period of from 0 to 42 or 43days in the rabbit when 5 mg of somatostatin is intramuscularlyadministered and/or

3. an AUC over a period of from 0 to 42 days of at least 160, preferablyof from 170-230 ng/ml×days, for the rat, when 10 mg of somatostatin issubcutaneously administered and/or an AUC over a period of from 0 to 42or 43 days of at least 160, preferably of from 180 to 275, e.g. from 200to 275 ng/ml×days for the rabbit, when 5 mg of somatostatin isintramuscularly administered.

For the quantitative characterization of the sustained releaseformulations described above we use the method of area deviation (AD)published by F. Nimmerfall and J. Rosenthaler; Intern. J. Pharmaceut.32, 1-6 (1986).

In brief, the AD method calculates the area deviations of theexperimental plasma profile from an ideal profile which is a constantaverage plasma level (=C_(p).ideal) produced by conversion of theexperimental area under the plasma level-time curve (AUC) to a rectangleof equal area. From the percental area deviation (referred to AUC) the Xretardation is calculated as follows:

retardation=100×(1-AD/AUC) By this method the whole plasma profilemeasured over a preselected time period is characterized by means of asingle numerical index.

In Proc. Natl. Acad. Sci. USA 85 (1988) 5688-5692 has been described inFIG. 4 a plasma level profile of the octapeptide analog of somatostatinof the formula

    D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Trp-NH.sub.2

in the rat.

However, a clear comparison can not be made with the plasma level dataof the compositions of the invention in the rat, mentioned just before,since the described plasma level profile was based on anotheradministration method (intramuscular injection) and--what is moreimportant--the microcapsules' loading level (between 2 and 6%) anddosage amount for administration (25 to 50 mg portions of microcapsulesfor 30 days, although for at least during 45 days determinations weremade) were not exactly indicated. Additionally the type of usedpoly(Dl-lactide-co-glycolide) was not exactly described.

The disclosure value of the publication is thus too low to admit it tobe a prepublication, interfering with the invention.

The following examples illustrate the invention.

M_(w) of polymers is the mean molecular weight as determined by GLPCusing polystyrene as standard.

EXAMPLE 1

One g. of poly(D,L-lactide-co-glycolide)(50/50 molar, M_(w) =45,000;polydispersity ca. 1.7) was dissolved in 15 ml of methylene chloridewith magnetic stirring followed by the addition of 75 mg of Octreotideacetate dissolved in 0.5 ml of methanol. Fifteen ml of silicon oil(brand Dow 360 Medical Fluid 1000 cs) (silicone fluid) was added to thepolymer-peptide mixture. The resulting mixture was added to a stirredemulsion containing 400 ml n-heptane, 100 ml pH 4 phosphate buffer, 40ml Dow 360 Medical Fluid, 350 cs and 2 ml Span 80 (emulsifier). Stirringwas continued for a minimum of 10 minutes. The resulting microparticleswere recovered by vacuum filtration and dried overnight in a vacuumoven. The yield was approximately 90% of microparticles in the 10 to 40micron size range.

The microparticles were suspended in a vehicle and administered IM in a4 mg dose of Octreotide to white New Zealand rabbits. Blood samples weretaken periodically, indicating plasma levels of 0.5 to 1.0 ng/ml for 30days as measured by Radioimmunoassay (RIA) analysis.

EXAMPLE 2

One g of poly(D,L-lactide-co-glycolide) glucose (M_(w) =45,000 (55/45molar produced according to the process of GB 2,145,422 B:polydispersity ca. 1.7; produced from 0.2% glucose) was dissolved in 25ml of ethyl acetate with magnetic stirring followed by the addition of75 mg of Octreotide dissolved in 3 ml of methanol. Twentyfive ml ofsilicon oil (brand Dow 360 Medical Fluid, 1000 cs) was added to thepolymer-peptide mixture. The resulting mixture was added to the emulsiondescribed in Example 1. Stirring was continued for a minimum of 10minutes. The resulting microparticles were recovered by vacuumfiltration and dried overnight in a vacuum oven. The yield was greaterthan 80% of microparticles in the 10 to 40 micron size range.

The microparticles were suspended in a vehicle and administered IM in a4 mg dose of octreotide to white New Zealand rabbits. Blood samples weretaken periodically indicating plasma levels of 0.5 to 2 ng/ml for 21days as measured by RIA.

EXAMPLE 3

A solution of 1.5 g of Octreotide acetate in 20 ml of methanol was addedwith stirring to a solution of 18.5 g ofpoly(D,L-lactide-co-glycolide)glucose (50:50 molar, Mw 45,000) in 500 mlof methylene chloride. Phase separation was effected by adding 500 ml ofDow 360 Medical Fluid (1000 cs) and 800 ml of Dow 360 Medical

Fluid (350 cs) to the peptide-polymer suspension. The resultant mixturewas added to a stirred emulsion consisting of 1800 ml of n-heptane, 2000ml of sterile water and 40 ml of Span 80. After stirring for 10 minutes,the microspheres were collected by vacuum filtration.

Half of the product was dried overnight in a vacuum oven at 37° C. Theresidual methylene chloride level was 1.2%.

The other half of the product was washed by stirring with 1000 ml ofethanol containing 1 ml of Span 80. After stirring for one hour, theethanol was decanted and the microparticles were stirred with 1000 ml ofn-heptane containing 1 ml of Span 80. After stirring for one hour, themicroparticles were collected by vacuum filtration and dried overnightin a vacuum oven at 37° C. The residual methylene chloride level of themicroparticles washed in this manner was reduced from 1.2% to 0.12%.

The combined yield of the product was 18.2 g (91%) of microparticlescontaining 5.6% Octreotide, mean diameter of 24 microns, 1.5% residualheptane.

The microparticles were suspended in a vehicle and injectedintramuscularly in 5 mg/kg dose of Octreotide to white rabbits. Bloodsamples were taken periodically, indicating plasma levels of 0.3 to 7.7ng/ml for 49 days as measured by RIA.

EXAMPLE 4

One g of poly (D,L,-lactide-co-glycolide)glucose M_(w) 46,000 (50:50)molar produced according to the process of GB 2,145,422 B,Polydispersity ca. 1.7, produced from 0.2% glucose) was dissolved in 10ml of methylene chloride with magnetic stirring followed by the additionof 75 mg of Octreotide dissolved in 0.133 ml of methanol. The mixturewas intensively mixed e.g. by means of an Ultra-Turax for one minute at20,000 rpm causing a suspension of very small crystals of Octreotide inthe polymer solution.

The suspension was sprayed by means of a high speed turbine (NiroAtomizer) and the small droplets dried in a stream of warm airgenerating microparticles. The microparticles were collected by a"zyklon" and dryed overnight at room temperature in a vacuum oven.

The microparticles were washed with 1/15 molar acetate buffer pH4.0during 5 minutes and dried again at room temperature in a vacuum oven.After 72 hours the microparticles were sieved (0.125 mm mesh size) toobtain the final product.

The microparticles were suspended in a vehicle and administered i.m. in5 mg/kg dose of Octreotide to white rabbits (chinchilla-bastard) ands.c. in a 10 mg/kg dose to male rats. Blood samples were takenperiodically, indicating plasma levels of 0.3 to 10.0 ng/ml (5 mg dose)in rabbits and 0.5 to 7.0 ng/ml in rats for 42 days as measured byRadioimmunoassay (RIA) analysis.

EXAMPLE 5

Microparticles were prepared by spray-drying in the same way asdescribed for example 4 with the only change that Octreotide wassuspended directly in the polymer solution, without use of methanol.

The microparticles were suspended in a vehicle and administered s.c. ina 10 mg/kg dose of Octreotide to male rats. Blood samples were takenperiodically, indicating plasma levels of 0.5 to 10.0 ng/ml in rats for42 days as measured by Radioimmunoassay (RIA) analysis.

EXAMPLE 6

One g of poly(D,L,-lactide-co-glycolide)glucose, M_(w) 46,000 (50:50molar produced according to the process of GB 2,145,422 B,Polydispersity ca. 1.7, produced from 0.2% glucose) was dissolved in 2.5ml of methylene chloride followed by the addition of 75 mg of Octreotidedissolved in 0.125 ml of deionized water. The mixture was intensivelymixed e.g. by means of an Ultra-Turax for one minute at 20,000 rpm(inner W/O-phase).

One g of Gelatine A was dissolved in 200 ml of deionized water at 50° C.and the solution cooled down to 20° C. (outer W-phase). The W/O- and theW-phases were intensively mixed. Thereby the inner W/O-phase wasseparated into small droplets which were dispersed homogenously in theouter W-phase. The resulting triple emulsion was slowly stirred for onehour. Hereby the methylene chloride was evaporated and the microcapsuleswere hardened from the droplets of the inner phase. After sedimentationof the microparticles the supernatant was sucked off and themicroparticles were recovered by vacuum filtration and rinsed with waterto eliminate gelatine.

Drying, sieving, washing and secondary drying of the microparticles wasdone as described for example 4.

The microparticles were suspended in a vehicle and administered i.m. in5 mg/kg dose of Octreotide to white rabbits (chinchilla-bastard) ands.c. in a 10 mg/kg dose to male rats. Blood samples were takenperiodically, indicating plasma levels of 0.3 to 15.0 ng/ml (5 mg dose)in rabbits and 0.5 to 8.0 ng/ml in rats for 42 days as measured byRadioimmunoassay (RIA) analysis.

EXAMPLE 7

Microparticles were prepared by the triple-emulsion technique in thesame way as desribed for example 6 with three changes:

1. 0.25 ml of acetate buffer pH 4.0 were used instead of 0.125 ml ofwater to prepare the inner W/O-phase.

2. rinsing after collection of the microparticles was carried out with1/45 molar acetate buffer pH 4.0 instead of water.

3. further washing of microparticles was omitted.

EXAMPLE 8

Microparticles were prepared by the triple-emulsion technique in thesame way as described for example 7 with the only change that the innerW/O-phase was prepared by using water containing 0.7% (w/v) sodiumchloride instead of acetate buffer.

EXAMPLE 9

Microparticles were prepared in the same manner as described in example6, with the only difference, that the drug compound is disperseddirectly in the polymer solution, whereafter the resulting dispersion ismixed with the gelatine containing water phase.

EXAMPLE 10 Octreotide Pamoate

10.19 g of octreotide free base (10 mM) and 3.88 embonoic acid (10 mM)are dissolved in 1 liter of water/dioxane (1:1). The reaction mixture isfiltered, and lyophilized to give a yellow powder α!²⁰ D=+7.5° (C=0.35,in DMF), of octreotide pamoate hydrate. Factor=1.4 wherein thefactor=weight of lyophilizate/weight of octreotide contained therein.

The pamoate may replace the octreotide acetate present in themicroparticles of Examples 1-9 and has an excellent stability.

EXAMPLE 11

A solution of 1 g of poly(D,L-lactide-co-glycolide) (50:50 molar,MW=36,100) in 20 ml of methylene chloride was added with stirring to asolution of 100 mg of calcitonin in 1.5 ml of methanol. Phase separationwas effected by adding 20 ml of silicone fluid (Dow 360 Medical Fluid,1000 cs). The resultant mixture was added to a stirred emulsionconsisting of 100 ml of pH 4 phosphate buffer, 400 ml of n-heptane, 4 mlof Span 80, and 40 ml of silicone fluid (Dow 360 Medical Fluid, 1000cs). After stirring for 10 minutes, the microspheres were collected byvacuum filtration and dried overnight in a vacuum oven at 37° C. Theyield was 1.1 g of microspheres containing 5.9% calcitonin.

EXAMPLE 12

A solution of 9.9 g of poly(D,L-lactide-co-glycolide) (50/50 molar,Mw=44,300) in 140 ml of methylene chloride was added to 100 mg oflypressin. The dispersion was magnetically stirred for one hour beforeadding 140 ml of silicone fluid (Dow 360 Medical fluid, 1000 cs) and 2.5ml of Span 80. The mixture was added to 1000 ml of heptane and stirredfor 10 minutes. The resulting microcapsules were collected by vacuumfiltration, washed three times with heptane, and dried 10 minutes undersuction. Half of the sample was washed by stirring in water for 10minutes; the other half was not washed. Both samples were driedovernight in a vacuum oven at 30° C. The total yield was 10.65 g ofmicrocapsules. Analysis of the washed sample was 0.5% lypressin and 0.6%for the sample not washed with water.

What is claimed is:
 1. A process for the production of microparticlescomprising ostreotide distributed throughout, which comprisesintensively mixing:a) a solution of octreotide in a weight/volume ratioof 2.5 g/10 ml in a buffer of pH 3-8 and b) a solution of apolylactide-co-glycolide in methylene chloride in a weight/volume ratioof 40 g/100 ml in such a manner that the weight/weight ratio of the drugto the polymer is 1/16 and the volume/volume ratio of the aqueousmedium/organic solvent is 1/10, intensively mixing the w/o-emulsion ofa) in b) together with c) an excess of a buffer of a pH 3-8, containinggelatine in a concentration of 0.5% of weight at a volume/volume mixingspeed ratio of ab)/c) of 1/40, without adding any drug retainingsubstance to the water-in-oil emulsion or applying any intermediateviscosity increasing step, hardening the embryonic microparticles in theformed w/o/w-emulsion by evaporation of the methylene chloride and byisolating, washing and drying the generated microparticles.